The present technology relates to Translational Regulatory (e.g. TR) nucleic acid molecules encoding mRNA molecules that are selectively translated and detected early in stressed and/or dying cells. In various embodiments, the technology relates to expression cassettes comprising such TR elements, mammalian cells and transgenic animals comprising such expression cassettes, and methods of use and treatment.
Normal biological activity in a living organism combines endogenous expression of genes that constitute an individual's genome with responses to the outside world. In higher eukaryotes, gene expression begins in the nucleus with transcription of genomic DNA into a pre-mRNA or “primary” RNA transcript. While still in the nucleus, the pre-mRNA is modified to include a 5′ cap structure, forms heteronuclear ribonucleoprotein (hnRNP) complexes, acquires a 3′ polyadenylate tail and undergoes splicing to remove intervening DNA sequences (e.g. introns). The mature mRNA is then exported to the cytoplasm where protein complexes direct (1) association with ribosomes via the 5′ cap structure, termed Cap-dependent translation, or (2) interaction with cytosolic RNA binding proteins that facilitate mRNA storage, processing or degradation. Following ribosome-driven translation, sequential shortening of the 3′-polyadenylate tail results in transport of the mRNA body to a complex of ribonucleases (RNAses), termed the exosome, which degrades the aged mRNA and effectively terminates protein synthesis.
As expected, gene expression is a highly regulated process that must produce a desired gene product (typically a polypeptide) at a particular time, rate and quantity. In addition to transcriptional regulation, post-transcriptional processes such as mRNA decay and translation are key checkpoints in gene expression. It is not surprising that changes in a cellular expression profile, produced by genetic mutations or aberrant responses to external stimuli can cause severe abnormalities that often result in cell death and the manifestation of a disease phenotype.
Extensive or prolonged cellular stimulation by environmental factors, such as altered nutrient levels, cytokines, hormones and temperature shifts, as well as environmental stresses like hypoxia, hypocalcemia, viral infection and tissue injury, results in the rapid attenuation of cap-dependent translation. This process is adaptive as it curtails the global synthesis of proteins which is not needed for an immediate stress response and recovery. However, this translational abatement does not completely eliminate ribosome activity, since many products of stress response and recovery genes continue to be synthesized by an alternative process, termed cap-independent translation (reviewed in Guhaniyogi & Brewer, 2001, Gene 265(1-2):11-23).
Cap-independent translation occurs by direct recruitment of ribosomes to specific RNA structures termed Internal Ribosome Entry Sites (IRESs). Bypassing the requirement for a 5′ mRNA cap structure was initially described as a mechanism for translating viral RNAs irrespective of a near complete inhibition of cellular cap-dependent translation in infected cells (Jang et al., 1988, J. Virol., 62:2636-43). Generally, IRES sequences cannot be identified by sequence homology and well characterized IRES elements have been verified using functional assays (Mountford and Smith, 1995, TIG, 11(5): 179-184; Baird et al., 2006, NAR, 12(10):1755-85). Current evidence shows that the conformation of the IRES RNA and the binding of accessory proteins to specific mRNA sequences enable ribosome binding. In eukaryotic cells, IRES-directed translation has often been associated with 5′ untranslated regions (5′UTRs) of mRNAs that contain unusually long and thermodynamically stable RNA secondary structures with multiple short open reading frames (ORFs) that dramatically inhibit the initiation of ribosome-dependent translation. However, functional verification of IRES activity for many of these 5′ UTR IRES elements has been complicated by the presence of transcriptional effector sequences cloned from the overlapping 5′ gene promoter. Attempts to employ these 5′UTR elements in IRES reporter vectors have been complicated by this residual background transcriptional activity which masks any translational regulation produced by these sequences.
IRES elements have been identified in a number of eukaryotic mRNAs (Bonnal S et al., (2003) Nucleic Acids Res. 31:427-428) and ensure the efficient expression of proteins or fragments thereof during nuclear inactivity or acute cellular stress when “cap-dependent” translation initiation is inhibited (i.e., apoptosis, starvation, gamma-irradiation, hypoxia, mitosis, or terminal differentiation). U.S. Published Patent Application No. 2006/0173168 discloses two low molecular peptides from the C-terminus of the PLP/DM20 gene, PIRP-M and PIRP-L, which are produced by internal translation initiation at an IRES.
The impact of chemical or biopharmaceutical intervention on the overall health of a specific individual is often uncertain. While a pharmaceutical molecule may remedy a targeted symptom, the treatment may be accompanied by serious side effects or unexpected toxicity that can, in some cases be worse than the initial malady. Although the side effects and toxicity of a pharmaceutical preparation are often known and may be limited to a small subset of individuals, these effects may be so severe in this small subset of individuals that a drug may not achieve FDA approval which results in huge pharmaceutical losses.
A large number of chemicals are manufactured in the United States annually. Over 2,000 new chemicals are introduced into the market each year, although very few are comprehensively tested for acute or chronic toxicity. In order to define the potential toxicity of a novel drug or chemical, the Food and Drug Administration (FDA) requires a New Drug Application (NDA) to include a large battery of toxicity, carcinogenicity, mutagenicity and reproductive/fertility tests in at least two animal species. The frequent, invasive testing and postmortem endpoint has raised considerable criticism from animal rights groups and the general public about animal suffering.
This situation underscores the need in the art for alternative, high-throughput molecular and biological screening technologies capable of detecting cell stress and toxicity in a broad spectrum of cell types following acute or chronic exposure to a chemical. Accordingly, novel methods for efficient and less expensive toxicity testing that provide a reliable alternative to animal testing are needed.